Abstract Lymphangioleiomyomatosis (LAM) is a progressive, destructive lung disease of women that can lead to oxygen dependency and death. LAM cells contain bi-allelic inactivating TSC2 gene mutations. The reasons for the female predominance of LAM and the mechanisms underlying cystic lung destruction are not well understood, representing key knowledge gaps that will be addressed in this proposal. The TSC1/TSC2 protein complex inhibits mTORC1. Multiple components of the TSC signaling network can localize to the lysosomal membrane, including mTOR, Rheb, TSC1, and TSC2. Lysosomes are highly dynamic organelles with both degradation and signaling functions, thus participating in many cellular pathways. TFEB is a ?master regulator? of lysosomal biogenesis and lysosomal exocytosis. We have found that TFEB is markedly elevated in the nucleus of TSC2-deficient cells and in human LAM cells. Lysosomal number is also increased in TSC2-deficient cells, and LAM cells have a striking increase in lysosomal proteins including NPC1. Lysosomal enzymes are released from the lysosomes through lysosomal exocytosis, which degrade extracellular matrix, and could be a cause of lung destruction in LAM. We have also discovered that estrogen strongly increases lysosomal gene expression in LAM patient-derived cells. Our central hypothesis is that elevated TFEB in LAM cells leads to increased lysosomal content and the release of lysosomal enzymes into the extracellular space, leading to lung destruction. We further hypothesize that these effects are enhanced by estrogen. A key translational corollary is that TFEB and/or lysosomal proteins are potential therapeutic targets for LAM. Our hypotheses will be tested in four Aims: Aim 1. To determine how TFEB impacts lysosomal exocytosis and the invasive potential of TSC2-deficient cells. Aim 2. To determine the mechanism through which estrogen affects lysosomal content and lysosomal exocytosis in TSC and LAM. Aim 3. To identify small molecules that inhibit the activity of TFEB in TSC and LAM. Aim 4. To determine how inhibition of TFEB and/or inhibition of lysosomal exocytosis impact lung destruction in a mouse model of LAM. We expect this project to have scientific and preclinical impact by elucidating the mechanisms through which LAM cells induce lung destruction and the reasons for the striking female predominance of LAM.